Hematological and physiological responses in polo ponies with different field-play positions during low-goal polo matches

Strenuous exercise in traditional polo matches creates enormous stress on horses. Hematological and physiological measures may vary across different field-play positions. This study aimed to investigate the effort intensity and the impact of exertion on hematology and heart rate variability (HRV) in polo ponies with different positions. Thirty-two ponies, divided equally into eight teams, were studied. Each comprises forwards (number 1), midfielders (numbers 2 and 3), and defenders (number 4). Team pairs played the first chukka in four low-goal polo matches. Percent maximum heart rate (%HRmax), indicating ponies’ effort intensity, was classified into five zones, including zones 1 (<70%), 2 (70–80%), 3 (80–90%), 4 (90–95%) and 5 (>95%). Hematological and HRV parameters were determined before, immediately after, and at 30-minute intervals for 180 minutes after chukkas; HRV variables were also obtained during warm-up and exercise periods. Results indicated that the number two ponies spent more time in zone 4 (p < 0.05) but less in zone 2 (p < 0.01) than the number four ponies. Cortisol levels increased immediately and 30 minutes afterward (p < 0.0001 for both) and then returned to baseline 60–90 minutes after exertion. Other measures (Hct, Hb, RBC, WBC, neutrophils, and CK enzyme) increased immediately (p < 0.0001 for all) and lasted at least 180 minutes after exertion (p < 0.05–0.0001). HRV decreased during the chukka until approximately 90 minutes afterward (p < 0.05–0.0001). The stress index increased during the chukka and declined to baseline at 60 minutes in number 1–3 ponies but lasted 90 minutes in those at number four. Effort intensity distribution differed among field-play positions. Decreased HRV indicated reduced parasympathetic activity during exercise, extending to 90 minutes after exertion in polo ponies. Defenders seem to experience more stress than those in other positions.

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Introduction
Horses are capable of high-intensity exercise because they can increase oxygen consumption, extraction, and cardiac output during explosive efforts such as escaping from predators [1].
However, horses utilized for sports may face appalling conditions involving exogenous and endogenous stressors during their career [1].Stress following involuntary activities in equestrian sports could lead to reduced performance, homeostasis disturbance, and medical problems in the body systems involved in those efforts [2,3].
Unlike other equestrian sports, polo is the only one where team pairs play against one another [4].Each team comprises four combinations in which the player's abilities are classified from -2 (low performance) to +10 (high performance).The total handicap of a team's players is summed to account for the team's handicap (goals) [4,5].Played on about a 10-acre (4.05 ha) grass field, a game consists of four, five, and six chukkas in low, medium, and highgoal competitions [6].Polo horses experience rapid speed acceleration, pauses, and swift direction changes throughout the 7 minutes of each chukka [4,5,7].This could lead to impaired homeostasis and potentially compromise horse welfare during the match, especially in unfit horses.
Increased red [4,5] and white blood cell [5] parameters have been observed immediately after polo matches.Furthermore, creatine kinase (CK) enzyme and lactate levels are also modulated [4,8,9], and blood pH and total carbon dioxide decrease, coinciding with increased base excess and lactate levels; these changes reflect that exertion during polo matches is at high-intensity [4,8].Measured heart rate (in terms of percentage of maximum heart rate, %HRmax) is frequently used to determine the cardiovascular demand and exercise intensity in sport horses in different disciplines [10,11], including polo ponies [7,12].One study showed that, during a low-goal polo match, ponies spent most of the time at less than 90% HRmax but a few minutes above 90%, suggesting that exercise during the match created moderate to high stress on the cardiovascular system [7].
It is well-accepted that levels of cortisol, a glucocorticoid hormone generated by the adrenal cortex, are frequently used to monitor stress levels in multiple conditions, such as road transportation [14,23], stress-related disease, [24] and during equestrian competition [13,25,26].It has been reported that horses participating in eventing experience higher cortisol levels than those in jumping and dressage [15].In contrast, endurance horses have the highest cortisol levels post-exercise, compared to the cross-country phase of 3-day events, trotting and galloping races, and jumping [27].Hence, stress levels differ among equestrian sporting disciplines according to exercise intensity and the demands of competing in specific sports [2,13,27].
As an alternative to cortisol, HRV has also been used as a non-invasive marker of autonomic and physiological responses that can reflect stress levels and welfare [13,14,22,28,29].HRV is a continuous measure of change in time differences between consecutive heartbeats during the cardiac cycle.Fluctuations in the interval between heartbeats are influenced by the interplay between sympathetic and parasympathetic (vagal) components that act on the sinoatrial node of the heart in conjunction with respiration and neurohormonal factors released into the blood circulation [30][31][32][33].The irregular time interval between sequential heartbeats benefits the adaptability and flexibility of the body to cope with difficult situations [29,33].HRV appears to be a reliable indicator of autonomic responses in horses in several conditions, including specific veterinary protocols [34,35], transportation [14,36,37], exercise training [20,22,38], and exercise during equestrian events [13,39,40].Several HRV measures indicate the sympathetic and parasympathetic (vagal) activities in response to external stimuli.
HRV time domains, including verified beat-to-beat (RR) interval and standard deviation of normal-to-normal RR intervals (SDNN), reflect the long-term heart rate variation affected by sympathetic and vagal activities [29,31].The root mean square of successive RR interval differences (RMSSD), the number of consecutive RR interval pairs that differ by more than 50 msec (NN50), and its relative value (pNN50) all point to the short-term heart rate variation under the influence of vagal activity [33].Triangular interpolation of normal-to-normal intervals (TINN) and the RR triangular index are geometric methods that express overall HRV during a measurement period [31,41].
HRV frequency domains are also computed to indicate sympathetic and vagal activities.
Modulation in the high-frequency (HF) band is affected mainly by vagal action, while the lowfrequency (LF) band changes under sympathetic and vagal components [29,31,33,42].
However, change in the very-low-frequency (VLF) band occurs following vagal activity, thermoregulation, renin-angiotensin, and vasomotor tone [31,43,44].Nonlinear results, consisting of the standard deviation of the Poincaré plot perpendicular to the line of identity (SD1) and the standard deviation of the Poincaré plot along the line of identity (SD2), reflect short-term and long-term HRV, respectively [29,33].Furthermore, SD1 and RMSSD have been reported to be identical in reflecting short-term HRV and vagal activity [45], while SD2/SD1 ratio indicates sympathovagal balance and correlates with LF/HF ratio [31,46].
Decreased RR interval, RMSSD, and SD1 reflect reduced vagal activity during short and medium-distance road transportation in horses [47].On the contrary, increased RMSSD following aromatherapy indicated a shift toward vagal action [19].Concerning equestrian competition, differences in RR interval, RMSSD, pNN50, SD1, and %VLF values were observed in horses jumping different fence heights [40].A reduction in RMSSD was also documented in horses participating in equestrian sports [13,21,48].In addition, the vast majority of HRV variables, including time domains (RR interval, SDNN, RMSSD, NN50, pNN50, TINN, and RR triangular index), frequency domains (VLF, LF, and HF bands) and nonlinear results (SD1 and SD2) decreased in horses during exercise [22,38].Hence, HRV is a helpful indicator for determining autonomic responses in horses.
Since the exercise pattern of equestrian polo [4] is similar to team sports such as soccer [49], knowledge of physical and physiological responses may be transferred from human team sports to monitoring bodily responses in polo ponies.It has been reported that wingbacks perform more maximum exercise intensity than forwards during competitive soccer matches [50].Moreover, from a young age, outfielders (forwards, midfielders, and defenders) displayed higher VO2max, mean aerobic power, and sprint performance than goalkeepers [51].Hence, different player positions experience distinct exercise intensities and physiological demands during soccer games.
Although there were reports on hematological and physiological responses in polo ponies participating in polo matches, responses in ponies across different field-play positions are scarce.Furthermore, the autonomic regulation of such ponies, particularly regarding their different positions during a match, has not yet been elucidated.Therefore, this study aimed to investigate the impact of traditional exercise regimens in polo on effort intensity distribution, cortisol levels, hematology, and autonomic regulation in ponies with different field-play positions.
They had been regularly trained according to the traditional polo training method [12] and playing two to four chukkas weekly during polo tournaments.They were housed separately in a 4 x 5 m stable within barns accommodating up to 40 horses.Daily feeding consisted of 2 kg of commercial pellets (provided across two meals) and 15 kg of hay hung permanently.Ponies had free access to tap water in their stables.They also spent approximately two hours daily in a paddock, except on match days.No ponies were undergoing medical or surgical treatment before the experiment commenced.The study protocol was approved by the Kasetsart University's Institute of Animal Care and Use Committee (ACKU65-VET-003).

Experimental protocol
The study was conducted in January 2022 during the Thai Polo and Equestrian Club tournament in Pattaya, Thailand.Thirty-two polo ponies were divided into eight teams, in which four player positions were deployed in a team, including number one (forwarder), numbers two and three (midfielders), and number four (defender).A team pair played against each other in four low-goal field plays outlined by the basic rules of polo [4].The player's handicaps within the teams were 8.88 ± 2.42 goals.Since there were four chukkas, with a period of six and a half minutes per chukka, the ponies were assigned to participate only in the first chukka of each match for four consecutive days.The matches occurred in the late afternoon (15.00-17.00h local time), during fine weather with an average humidity of 37.75 ± 2.75% and mean temperature of 35.60 ± 0.57 ºC.The relevant sampling was conducted before, during, and after the first chukka.

Hormonal and hematological analyses
Hormonal and hematological variables were determined before the chukka, immediately afterward, and at 30-minute intervals for a further 180 minutes.In brief, 9 ml of whole blood was withdrawn from the jugular vein and divided into two parts.Three ml of blood was placed in potassium EDTA tubes for evaluating hematocrit (Hct), red blood cells (RBC), hemoglobin concentration (Hgb), white blood cells (WBC), neutrophils, and lymphocytes using an automated hematology analyzer (Advia®2120i; Siemens Healthineers, Erlangen, Germany).
Another portion of the blood sample was placed in 6 ml clotting activator tubes to obtain serum for biochemical examination, including 1) cortisol level determination using a competitive chemiluminescent enzyme immunoassay (IMMULITE Analyzers, Siemens Healthineers, Erlangen, Germany) and expressed as nmol/L, and 2) evaluation of creatine kinase (CK) level using a Liquid NAC activated UV test (HUMAN Gesellschaft für Biochemica und Diagnostica mbH, Wiesbaden, Germany) and expressed as ukat/L.

Data collection
Ponies were equipped with portable Polar heart rate monitoring (HRM) device sets (Polar Electro Oy, Kempele, Finland) for HR and HRV analyses.The device set comprises a Polar heart rate equine belt for trotters, a heart rate sensor (Polar H10), and a sports watch for recording RR intervals (Polar vantage 2).In brief, the heart rate sensor was attached to the equine belt for trotters at the sensor pocket on the belt and then soaked in water to increase signal transmission.The soaked belt was fastened around the pony's chest, where the sensor was placed on the left side of the chest.The sensor was connected to the sports watch to record RR interval data 15 minutes before the chukka.The players wore sports watches for recording RR intervals during the competition, which were then left close to the ponies with whom the sensors were paired for 180 minutes after the competition.
to correct artifacts and ectopic beats in the interbeat interval (IBI) data; in turn, this produces more accurate HRV variables than the standard version [52].The Kubios HRV Scientific software also supports automatic noise detection to identify noise segments that distort various consecutive beat detections in the IBI data.The automatic noise detection was set at a medium level in this study.Before analyzing HRV variables, smoothness priors were used to remove IBI time series nonstationarities.According to the user guideline manual, the cutoff frequency was fixed at 0.035 Hz (https://www.kubios.com/downloads/Kubios_HRV).
The HRV variables were as follows: Time domain results: RR interval, HR, SDNN, RMSSD, pNN50, TINN, RR triangular index, stress index, heart rate deceleration capacity computed as a four-point difference (DC) index, and modified DC computed as a two-point difference (DCmod).
The HRV variables were determined 15 minutes before the match began, during the six and a half minutes of the first chukka, and at 30-minute intervals for 180 minutes after it.

Speed and distance covered
The ponies' riding speed and distance covered were recorded by the Polar sports watches worn on the players' wrists throughout the chukka and exported as km/h and kilometers, respectively.

Statistical analysis
HR and HRV data were analyzed using GraphPad Prism version 10.1.0(GraphPad Software Inc, San Diego, USA).In an analysis of two-grouping variables, two-way ANOVA with repeated measures was applied to evaluate the independent effects of field-play position and time, as well as the interaction effect of field-play position and time, on changes in cortisol level, hematological, HR, and HRV variables in response to the exertion during competition.
Dunnett's post-hoc test was later implemented to assess between-group differences at given time points and within-group differences compared to the control (baseline values taken before competition).Tukey's post-hoc test was also applied when necessary.Two-way ANOVA was further used to determine the independent effects of field-play position and effort intensity zone (%HRmax) and their interaction on the time-spent distribution within zones.Tukey's post-hoc test was implemented to compare time spent in different effort zones within and between fieldplay positions.
Regarding the one-grouping variable analysis, the data's normal distribution was verified by the Shapiro-Wilk test.Due to the data being normally distributed, an ordinary one-way ANOVA followed by Tukey's post-hoc test was employed to estimate differences in riding speed and covered distance among the ponies playing in different positions.The Kruskal-Wallis test, accompanied by Dunn's multiple comparisons tests, was alternatively applied to assess players' handicaps among different field-play positions because the data showed a non-normal distribution.Data were expressed as mean ± SD, and p < 0.05 was considered statistically significant.

Player's handicap, riding speed, and distance covered
The average handicap of the number four players was higher than number one players (3.75 ± 1.39 vs. 1.38 ± 1.06, p = 0.0170) (Fig. 1a).The average riding speed and covered distance did not differ among the field play positions (Figs.1b and c).

Time spent within different effort zones
There was an interaction between field-play position and effort intensity zone (%HRmax) (p = 0.0497) and the separate effect of effort intensity zone (p < 0.0001) on the distribution of time spent within the zones.The ponies at the number one position spent more time in zone 3 compared to zones 1 (p < 0.05), 4 (p < 0.05) and 5 (p < 0.01).The ponies at number two position spent more time in zone 3 than in zones 2 and 5 (p < 0.05 for both), and in zone 4 more than zone 5 (p < 0.01).The time spent among different zones by number three ponies did not vary.
The number four ponies spent more time in zones 2 and 3 than both zone 4 (p < 0.05 for both) and zone 5 (p < 0.001 for both).The number four ponies spent more time in zone 2 (p < 0.01), and less time in zone 4 (p < 0.01), compared with number two ponies (Fig. 2).

Hormonal and hematological analyses
Since there was no interaction effect between group and time on changes in hormonal levels and hematological parameters, the parameters were subsequently evaluated as a pool, following the main effect of time.Cortisol levels increased immediately after the match and remained high until 30 minutes afterward (p < 0.0001 for both periods).After reducing to the baseline value at 60-90 minutes after the match, cortisol levels decreased to below the baseline at 120-180 minutes (p < 0.0001 for all periods) (Table 1).
At the same time, hematological parameters (including Hct, Hb, and RBC) increased immediately until 180 minutes afterward (p < 0.0001 for given periods of all variables).Even though WBC and neutrophil levels rose immediately until 180 minutes after competition (p < 0.0001, except for p < 0.05 at 60 minutes after competition for both variables), lymphocytes were unchanged throughout the study period (Table 1).

Time domain results
Only time exerted effects on HR and most time domain HRV variables, except for stress index, which demonstrated a group-by-time interaction (p = 0.0322), in addition to the independent effect of time.
The minimum HR increased during the warm-up and competition period (p < 0.0001 for both periods).Despite a gradual reduction, the minimum HR remained higher than the baseline values until 180 minutes after the competition (p < 0.0001 for all periods) (Fig. 3a).
The maximum HR also rose dramatically during the warm-up and peaked during the competition (p < 0.0001 for both periods).It was still high 30 minutes after the competition (p < 0.0001) and then reduced considerably to the baseline 60-90 minutes afterward.Maximum As expected, mean HR increased during the warm-up and competition (p < 0.0001 for both periods).After that, it reduced dramatically 30 minutes after the competition but remained higher than the baseline (p < 0.0001).Although it continued to decrease, it remained higher than the baseline 180 minutes after the match (p < 0.05) (Fig. 3c).In contrast to HR variables, the mean RR interval decreased during warm-up (p < 0.00001) and plunged to its lowest value during competition (p < 0.0001).There was a sharp rise in mean RR intervals 30 minutes (p < 0.0001) after the match.However, they then reached a plateau and remained higher than the baseline 60-180 minutes after the competition (p < 0.01-0.001)(Fig. 3d).SDNN, RMSSD, and pNN50 shared the same trend: they dropped dramatically during warm-up (p < 0.0001 for all variables), then continued to decrease to their lowest values during the competition (p < 0.0001 for all variables).After competition, they increased markedly 30-90 minutes (p < 0.001-0.0001for all variables) until reaching the baseline at 120 minutes.In addition, the pNN50 was higher than the baseline at 180 minutes after the match (p < 0.05) (Figs.4a-c).
Since an interaction was observed between field-play position and time, the stress index differed among the ponies in the match.The stress index increased during warm-up (number one ponies, p < 0.01; number two, p < 0.05; number three, p < 0.001; number four, p < 0.01) and peaked during competition periods (p < 0.0001 for number one, two and three ponies and p < 0.001 for number four ponies).It then reduced sharply 30 minutes after competition (number one ponies, p < 0.001; number two, p < 0.05; number three, p < 0.01; number four, p < 0.001).Although the stress index decreased to the baseline 60 minutes after competition in the ponies at numbers 1-3, it later reduced to the baseline 120 minutes after competition in those at number four (p < 0.01) (Fig. 4d).TINN and RR triangular index decreased during warm-up (p < 0.0001 for both variables) and were lowest during the competition (p < 0.0001 for both) (Figs.5a and b).TINN increased 30 minutes after the match (p < 0.0001) and returned to the baseline 60 minutes later (Fig. 5a).In contrast, the RR triangular index was higher at 30-90 minutes (p < 0.05-0.0001)and reached the baseline 120 minutes after the match.However, the RR triangular index rose further and was higher than the baseline 150-180 minutes afterward (p < 0.01-0.001)(Fig. 5b).
DC and DCmod reduced during warm-up, with the lowest values during the match (p < 0.0001 in both periods of the two variables).They increased 30-90 minutes after the match (DC, p < 0.01-0.0001;DCmod, p < 0.001-0.0001)and reached the baseline 120 minutes later (Figs.5c     and d).

Nonlinear results
SD1 and SD2 also decreased during warm-up and the match (p < 0.0001 for both periods of the two variables).They increased 30-90 minutes after the match (p < 0.001-0.0001for both periods of the two variables) and reached the baseline 120 minutes afterward (Figs.8b and c).
An increase in SD2/SD1 was observed during the warm-up and competition (p < 0.0001 for both periods) (Fig. 8d).

ANS indexes
SD1% decreased, corresponding to an increase in SD2% during warm-up and competition (p < 0.0001 for both periods) (Figs.9a and b).The PNS index declined after the warm-up and plunged to its lowest value during the match (p < 0.0001 for both periods).It then rose 30-120 minutes after it (p < 0.05-0.0001)and returned to the baseline by 150 minutes afterward (Fig. 9c).The SNS index increased during the warm-up and peaked during the match (p < 0.0001 for both periods).A sharp decrease was detected 30 minutes after (p < 0.0001), then gradually reduced between 60-90 minutes after the match (p < 0.001-0.0001)(Fig 9d).

Discussion
The present study determined the impact of a classic exercise regimen in a polo match on effort distribution, cortisol release, hematology, and heart rate variability.The significant findings from this study were: 1) number two ponies spent more time exercising in effort intensity zone 4, but less in zone 2, than number four ponies.2) There were no differences in cortisol levels and hematological modulation among ponies with different field-play positions.3) Cortisol levels rose considerably during competition and returned to the baseline 60 minutes later.4) Hct, RBC, Hb, WBC, neutrophils, and CK levels increased immediately and remained higher than the baseline up to 180 minutes after exertion.5) Mean HR increased considerably and remained higher than the baseline at 180 minutes.6) HRV modulation did not differ among ponies in different field-play positions, except for the stress index, which declined later in the number four ponies than those in other field-play positions.7) HRV decreased to the lowest values during exercise, gradually increasing and returning to baseline 120 minutes afterward.
The results suggest that polo ponies at different field-play positions experienced different levels of effort intensity.A decreased HRV reflected a shift toward sympathetic dominance during warm-up, with a substantial sympathetic effect during the workout, which lasted for approximately 90 minutes afterward.The number four ponies appeared to experience more stress than those in other positions, as indicated by the stress index modification.To our knowledge, this is the first study to report the physiological demands and autonomic responses in polo ponies with different field-play positions in low-goal polo matches.
In soccer, players' physical and physiological demands also vary across different positions [50,51,53].Wingbacks spend more time in higher-intensity zones than forwards [50].Wingbacks typically must participate in offensive action along with the forwards, alternating with a sprint back to cover opponents in a defensive system.In contrast, forwards and defenders spent more time with lower exercise intensity than other player positions because their specific task was scoring [50].It is worth noting that specific training regimens, with appropriately adjusted workloads, can increase players' performance in different positions.
Since equestrian polo and soccer have similar exercise patterns, differences in the positional demands are also expected in this equestrian sport.
In polo, each field-play position serves a distinct role.The number one position is usually filled by a player with relatively low experience, who is mainly responsible for scoring and neutralizing the opposite number four.In contrast, the number two player acts as a scrambler who needs a keen eye and fast ponies to scrap for the ball.They may feed the ball to the number one player or score alone.The number three player is the primary tactical player who provides the ball to the number one and two players, while the number four plays a crucial role in the defensive system to prevent the opposition from scoring.In this study, the number two ponies spent more time in the higher effort zone and less time in the lower effort zones than those at number four.This seems consistent with the multifunctional role of the number two player [6].In contrast, spending more time in lower effort zones in number four ponies may be consistent with their primary role in defense, undergoing multiple changes in direction and speed to prevent scoring; consequently, the running speed of number four ponies may not reach the high values observed in number two ponies.Our data thus suggest that ponies' physical effort within the chukka varies among different field-play positions and that specific training may be required for each position to reach the required performance at the respective intensity levels.
Serum cortisol levels and hematological parameters changed during the matches, regardless of field-play positions.Cortisol levels are known to be modulated in horses participating in various equestrian sports [13,26,27], including polo [5,54].The modulation we observed was consistent with previous studies in horses [55,56] and polo ponies specifically [5], showing a sharp increase in serum cortisol immediately, lasting 30 minutes, and returning to baseline 60 minutes after exertion.These results reflect the enormous stress on the ponies during high-intensity exercise in the chukkas.However, a further decrease in cortisol levels (below the baseline) at 120-180 minutes may result from the hormone's circadian rhythm during the late evening [22,[57][58][59][60].
The observed increase in CK enzyme levels parallels those of a previous report [5], demonstrating a rise in after the chukkas, but within the normal reference range of the enzyme.
Increased CK levels are believed to accompany high-intensity exercise due to muscle microtrauma, leading to increased fiber membrane permeability, as reported elsewhere [49,61].Increases in Hct, Hb, RBC, and WBC after exertion were also observed, corresponding to previous studies [4,5].It is well-established that red blood cell parameters rise following splenic contraction to increase oxygen-carrying capacity during intense exercise [4,5,62,63].
Furthermore, increased WBC levels are thought to be due to a shift into the bloodstream of lymphocyte [64] and neutrophil subpopulations [65] from the spleen, bone marrow, and lymph nodes.These reactions are influenced by cortisol and catecholamine releases following physical effort [66,67].
Although the enhancement of WBC levels immediately after chukkas was similar to changes reported by Zobba et al. [5], a disparity in leucocyte modulation and distribution was detected compared to their study.They demonstrated that a transient rise in WBC was observed immediately after the chukka [5], compared to a more prolonged increased WBC count immediately and until 180 minutes after the chukka in our study.Moreover, the presence of increased lymphocytes, but not neutrophils, in polo ponies of the previous study [5] contrasted with our findings, which showed increased neutrophils instead of lymphocytes.Since a change in leukocyte distribution and magnitude of leucocytosis are related to exercise intensity and duration [68,69], a difference in effort intensities (indicated by HR during the match) between the previous (~80 beats/min) and current (~175 beats/min) studies may be the underlying reason for the discrepancy.Although the ponies in this study showed increased serum cortisol levels and hematological profile after the chukkas, all parameter changes were within the normal reference range [5,70].These laboratory results may reflect the ponies' capability for physiological adjustment during physical effort in polo matches.
Concerning autonomic regulation during exertion, HRV typically decreases during conventional exercise in distinct equestrian sports [13,39,40,71].A lower HRV indicates a synchronous increase in sympathetic tone and decreased vagal tone or independent action of increased sympathetic or reduced vagal activities [29,33].It has been reported that RMSSD decreased during competitive exercise but did not differ between horses in jumping and dressage events [13].However, a difference in autonomic responses to ultra-short-term stimuli was observed in sports horses.and within the chukka.These reductions in HRV were consistent with previous reports describing HRV modulation in response to exercise [22,38], mirroring a decreased role of the vagal component and a shift toward sympathetic activity during exercise in polo.More importantly, higher HR indicated greater effort intensity during the chukka than during warmup period.The increased effort intensity corresponded to a further decrease in HRV variables and, in turn, a progressive decrease in vagal activity during the chukka.These results indicated that exercise intensity was a contributing factor to HRV modification.Even though the frequency domain variables (VLF, LF, HF, and total power bands) decreased during the warmup and the chukka, the contribution of each power spectrum band to the total power spectrum during the chukka differed from other given periods in the study.
LF/total power ratio remained unchanged throughout the study.In contrast, VLF/total power ratio increased, coinciding with a decreased HF/total power ratio during exercise.These results indicated a major contribution of the VLF band, corresponding to a decreased HF band, to the total power spectrum during exercise.Since the VLF band is modulated in response to vasomotor tone, thermoregulation, and renin-angiotensin action [72], an increased proportion of VLF contribution may, at least in part, point out the apparent effect of vasomotor tone thermoregulation and renin-angiotensin action, along with sympathetic dominance, on autonomic regulation during the chukka.An increase in LF/HF ratio, SD2/SD1 ratios, SNS index, and SD2%, coinciding with reduced PNS index and SD1%, provided supporting evidence of an increased sympathetic and decreased vagal activity during exertion.Despite no differences in the HRV variable, the stress index differed among field-played positions.The stress index was reduced later among ponies in the defensive position than those playing in other positions, suggesting that defenders tend to be under more stress.This observation is partly consistent with a previous study reporting that defenders had the highest anaerobic power utilization, leading to reduced blood pH compared with those in other positions [8].
Accordingly, more care may be taken in selecting ponies for defensive positions to avoid compromised horse welfare in equestrian polo.
Although we report differences in hormonal, hematological, and autonomic responses according to the positions in which ponies compete in the first chukka of a low-goal match, the question arises as to what extent such modulations would occur in ponies competing in more than one chukka.In addition, the effect of age on the effort intensity distribution and HRV modification in such ponies needs further investigation.This study's main limitation was that various players rode the ponies without controlling for their weight.The potential impact of different loading on physiological responses in polo ponies during the games may be considered in the future.

Conclusion
The effort intensity distribution differed among polo ponies in different field-play positions during the chukka.Blood cortisol levels and hematological parameters did not vary with position.However, cortisol levels increased immediately and 30 minutes after the match.In work included in this submission.Review the submission guidelines for detailed requirements.View published research articles from PLOS ONE for specific examples.
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Fig. 4 .
Fig. 4. Time domain analysis of HRV in response to exercise in polo matches, including

Fig. 9 .P- 2 ;
Fig. 9. Autonomic nervous system index, including SD1% (a), SD2% (b), parasympathetic contrast, red and white blood cell variables (except lymphocytes) increased immediately to 180 minutes after matches, while CK enzyme levels rose until 150 minutes afterward.A marked decrease in HRV indicated a shift toward sympathetic activity and physiological stress during warm-up and competition until 90 minutes afterward.The increased stress index of ponies at the number 4 position lasted longer than the other positions, implying that ponies in the defensive position tended to experience more stress than other positions.These results provide insight into effort intensity distribution, hematology, and autonomic regulation in response to exertion in polo ponies at different field-play positions.The information may, at least in part, be beneficial for selecting appropriate polo ponies regarding adequate fitness and proper autonomic function or adding specific training programs to reach appropriate fitness levels for typical field-play positions in polo, especially the number 2 and 4 ponies.More importantly, compared to before the game, the finding that expression of sympathetic dominance lasts 90 minutes after the match could heighten awareness of the prolonged physiological stress in polo ponies, despite playing only one chukka.

Table 1 .
Biochemical and hematological parameters (mean ± SD) in polo ponies (n=32 ) before and after low-goal polo competition.
a, b and d indicate statistical differences of given time points compared to before competition values at p < 0.05, p < 0.01, and p < 0.0001, respectively.
Villas-Boas et al. (2022) demonstrated a lower LF/HF ratioindicating a minimal autonomic response to the startle test-in endurance horses compared with dressage and polo horses [54].It was suggested that autonomic response to startling challenges, but not exercise, differed among horses in different fields.In this study, a decrease in HRV variables derived by time domain (SDNN, RMSSD, pNN50, TINN, RR triangular index, DC, and DCmod), frequency domain (VLF, LF, and HF bands), and nonlinear (SD1 and SD2) methods was detected, regardless of field play positions, during exercise in the warm-up